Membrane proteins specific to cold acclimation were studied in alfalfa (Medicago falcata L. cv Anik) seedlings and cell cultures. They were identified following separation on polyacrylamide gels and localized as far as possible to specific membranes by fractionation on continuous sucrose gradient and analysis of marker enzyme assays. / With cold acclimation, certain membrane proteins from seedlings showed subtle changes (mainly increases) in their steady-state amount and in their net synthesis rate. Most of these changes were in proteins with molecular weights below 100 kDa and were associated with light fractions of the sucrose gradient, favoring a Golgi, endoplasmic reticulum or tonoplast location for the proteins. Preliminary work done on membrane proteins from cell cultures showed more pronounced changes with cold acclimation. Most of the changes were in proteins with molecular weights below 100 kDa and were associated with heavy fractions of the sucrose gradient. Since they are easier to harvest and to manipulate, cell cultures appear to be the better system to use in future studies.

Infectious bursal disease virus (IBDV) is a chicken pathogen that infects the bursa of Fabricius, an organ involved in the development of the immune system in chickens. Infection by the virus leads to destruction of the bursa and immunosuppression. Infection by virulent strains may result in mortality. Current methods to combat the virus involve the use of vaccines. These are usually a mixture of live attenuated and oil inactivated virus. Variant strains of the virus are able to escape the vaccine-generated antibodies. In addition, the vaccines result in damage to the bursa. Identification of a receptor for IBDV could result in the development of either treatment for the virus or superior vaccines by interfering with the attachment of the virus to host cells. Several methods for identifying IBDV binding proteins from the membranes of cells from the bursa of Fabricius were examined. Affinity chromatography of IBDV binding proteins with a matrix consisting of IBDV cross-linked to Sepharose 4B allowed separation of a number of virus binding proteins. In contrast, virus overlay protein blot assay (VOPBA) and reversible cross-linking with 2-iminothiolane proved less; conclusive. Predominant proteins in the affinity-separated fraction were of 40 and 32 kDa. These were further examined by N-terminal amino acid sequencing of the whole protein and N-terminal sequencing of peptides produced by endoproteinase Lys-C digestion of the protein respectively. The 40 kDa protein showed homology with human synovial stimulatory protein involved in the formation of autoantibodies in rheumatoid arthritis. Virus was also shown to bind to a 440 kDa protein complex. This 440 kDa protein complex appeared to consist primarily of a 40 kDa protein when examined by reducing Tris-Tricine SDS-PAGE. Analysis of bursal membrane proteins by Western blots using sera from rheumatoid arthritis patients revealed interactions between several IBDV proteins and the antibodies from rheumatoid arthritis patients. Using serum from one of the five patients showed a strong interaction at approximately 80 kDa and a weaker interaction at approximately 40 kDa. This may indicate an immune reaction between a chicken homolog of the synovial stimulatory protein and antibodies in rheumatoid arthritis sera. The 32 kDa protein showed homology to a Pseudomonas fluorescens protein. A section of this sequence was amplified by PCR from chicken DNA and RT-PCR from chicken RNA using degenerate primers constructed from the established N-terminal amino acid sequences and chicken codon usage tables. The fragment produced upon amplification from chicken DNA and RNA did not correspond to the predicted size of 177 bp. In contrast, when the RT-PCR product was heated and snap cooled before examination by agarose gel electrophoresis, the product consisted of two fragments, one of approximately 400 bp in size and one of approximately 200 bp in size. The establishment ofthe 40 and 32 kDa chicken bursal membrane proteins as possible receptors for the virus could allow for the development of vaccines and/or treatment strategies for the virus. Treatment strategies or vaccines would be based on blocking of the interaction between IBDV and chicken host cells. Peptide mimics of the epitopes involved in such interactions could possibly achieve this. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Technological developments have enabled mass spectrometry (MS) to evolve asone of the most versatile, sensitive and widely used analytical methods. Key areas ofresearch in biological MS include the development of analyte-selective MSmethodologies, along with the design of MS compatible separation technology. Analytesof interest range from small, biologically active molecules in disease progressionresearch, to macromolecules such as proteins, in proteomics investigations. Advances inthese areas are vital to maintaining the level of sophistication that has become thebenchmark for MS analyses.Mass spectrometry has found a permanent station in disease progression studies,particularly in biomarker discovery. This is especially true for Alzheimer's disease (AD),a condition marked by widespread lipid peroxidation (LPO) in the brain. The mainhypothesis of the first part of this dissertation is that LPO produces aldehydes that canpotentially be exploited as AD biomarkers. Design of novel LC-MS/MS methods forbrain aldehyde analysis is described. The methods were applied towards aldehydequantification in the hippocampus, superior and middle temporal gyrus and cerebellum ofsubjects with early AD (EAD), mild cognitive impairment (MCI) and age-matchedcontrols. Results obtained indicated elevation of neurotoxic aldehydes in MCI and EADbrain and suggested that LPO occurred early in AD. Understanding AD progression hasbecome important for developing diagnostic methods and treatments.Mass spectrometry is also the major analytical tool in proteomics, where gelelectrophoresis is dominant in pre-MS separations. The main hypothesis of the latter partof this dissertation is that exposure of microbe fermenters including Clostridiumthermocellum to an external stimulus, such as ethanol, can alter the membrane proteome.Design of novel doubled-SDS-PAGE (dSDS-PAGE) methods for membrane proteinanalysis is described, as these proteins are under-represented in standard 2D-PAGE. Thenewly developed Bicine-dSDS-PAGE offered superior separation over other methods andwas applied towards analysis of wild type and ethanol-adapted C. thermocellum cellmembranes. Significant differences in protein expression were observed. Anunderstanding of ethanol adaptation will promote the design of more ethanol-tolerantstrains. Such an outcome can have dramatic effects in the fuel industry as the trendtowards more efficient fuel development gathers momentum.

The proteins and molecular machinery mediating the release of cortical granule (CG) contents from fertilized embryos is not completely understood. The process of vesicle fusion involves linking chaperones prior to vesicle to membrane contact. Rab3A, a member of a low-molecular weight GTP-binding protein superfamily has been detected in mouse embryos from the unfertilized meiotic II stage to the 2-cell. It is believed to positively regulate the final step of CG exocytosis by binding to Rabphillin, calcium ions (Ca2+), and phospholipids. SNAP23 a member of soluble NSF [N-ethylmaleimidesensitive factor] attachment protein receptors (SNAREs) binds together with parts of the Rab3A-rabphilin3A complex and is believed to be involved in the Ca2+-dependent exocytosis of non-neuronal systems. In this study we observed the mRNA expression for SNAP23 and Rab3A in pre-Meiotic I, post-Meiotic I unfertilized eggs (pre-MI UFE and post-MI UFE), and fertilized eggs (FE) utilizing RT-PCR. The products were analyzed in 2% agarose gel stained with ethidium bromide. Density analysis using a globin external standard showed that the levels of mRNA transcripts declined from the UFE to the FE in both genes, SNAP23 and Rab3A. Immunofluorescence was used for the detection and localization of Rab3A protein within the pre-MI and post-MI UFE and FE mouse egg. Eggs were stained with anti-Rab3A primary antibody and lens culinaris agglutinin (LCA) conjugated to FITC. Rab3A showed punctate staining in pre- and post-MI UFEs on small vesicles assumed to be CGs and in FEs on vesicles of a larger size. Uniform cytoplasmic expression was also seen, throughout the cells cortical and subcortical regions in each stage (pre- and post-MI UFEs and FEs), but with decreasing intensity as the eggs matured. This cytoplasmic stain may represent inactive Rab3A in the cytosol. The LCA stain showed punctate expression of cortical granules with localization within the cortical region and the plasma membrane. The addition of information on SNAP23 and Rab3A will aid in the process of studying CG exocytosis as well as in understanding the temporal and spatial development pathways involved in stimulating the cortical reaction. / Department of Biology

Off-resonance unenhanced total internal reflection (TIR) Raman Spectroscopy was explored to investigate supported single lipid bilayers with incorporated membrane peptides/proteins at water/solid interface. A model membrane was formed on a planar supported lipid layer (pslb) by the fusion of the reconstituted small unilamellar vesicles (SUVs), and the intensity of bilayer was confirmed by a comparison of Raman spectral intensity in the C-H stretching modes with C₁₆TAB. With prominent Raman sensitivity attained, we studied the 2-D phase transition of DMPC and DPPC pslbs and the temperature-dependent polarised spectra revealed a broad transition range of ca. 10 °C commencing at the calorimetric phase transition temperature. We applied polarised TIR-Raman Spectroscopy to pslbs formed by DMPC SUVs reconstituted with a model membrane-spanning peptide gramicidin D. A preferential channel structure formed by dissolution of trifluoroethanol could be probed by polarised Raman Spectroscopy qualitatively showing an antiparallel β-sheet conformation (different from "standard" one) and our Raman spectra by correlation with NMR and CD data confirmed single-stranded π^6.3 β-helical channel structure in the single bilayer. We also studied the membrane-penetrating peptide indolicidin in the presence of DMPC pslb over the chain melting temperature and a β-turn structure was dominantly observed concomitant with membrane perturbation. Dynamic adsorption of DPPC to form pslb from a micellar solution of n-dodecyl-β- _D-maltoside could be examined with high sensitivity of every 1-min acquisition. Finally we used polarised TIR-Raman scattering to porcine pancreatic phospholipase A₂ hydrolytic activity on DPPC pslbs and revealed lipid-active conformation different from that of the enzyme alone.

No description available.

Despite being amongst the most common zoonotic diseases in the world, brucellosis is a neglected disease for which an approved vaccine for human use does not exist. Thus far, the traditional approaches to Brucella antigen selection for subunit vaccine development have yielded unacceptable results. In this work, we evaluated the predictive ability of a multistep Brucella antigen selection process with in vitro immunological and invasion assays and in vivo protection experiments. Initial in silico screening for antigens was performed via genomic sequence analysis where 27 Brucella melitensis open reading frames (ORF) coding for outer membrane proteins bearing MHC epitopes, adhesin and conserved properties were identified. Evidence for a role in any aspect of Brucella virulence (i.e., invasion, co-regulation/expression with known Brucella virulence factors, intracellular adaptation) was then used to narrow the list of candidate antigens. To further increase confidence in the candidate ORF putative role in Brucella pathogenesis, differential expression of candidate ORF was evaluated using previously generated global transcriptomics data in in vitro HeLa and in vivo bovine models of acute Brucella infection. Protein expression in the E. coli heterologous system resulted in the successful expression of OmpW, BtuB, Omp22, Hia, and FlgK. With regards to virulence, the two proteins with the highest predicted adhesin scores conferred an invasive phenotype to the non-invasive BL-21 E. coli strain in alveolar epithelial cells. From an immunogenicity standpoint, all proteins elicited IgG production in Brucella-exposed goats, mouse and humans. Antigen-specific recall responses in splenocytes from C57BL/6 mice immunized with a cocktail of the three proteins with highest MHC scores revealed a mixed Th1/Th2 response with a comparatively greater Th1 response. In protection studies, subcutaneous (SQ) immunization with BtuB, Hia and FlgK, individually, promoted bacterial clearance following a robust intraperitoneal challenge dose of Brucella melitensis 16M. In addition, single SQ inoculation of FlgK enhanced protective efficacy of the vaccine strain B. abortus S19. In contrast, immunization of mice with the three protective antigens in a cocktail formulation elicited immune responses but no protection against intraperitoneal challenge with Brucella melitensis 16M in the spleen and liver. In conclusion, our results indicate that our combinatorial in silico, in vitro and in vivo antigen selection and identification modeling approach provides strong evidence for prediction of Brucella protective antigens, and represent a novel strategy with broad application to other major pathogens.

Membrane proteins are found in all kingdoms of life and are essential for cellular interactions with the environment. Although a large research effort have been put into this group many membrane proteins remains uncharacterized, both in terms of function and evolutionary history. We have estimated the component of α-helical membrane proteins within the human proteome; the membrane proteome. We found that the human membrane proteome make up 27% of all protein, which we could classify the majority of into 234 families and further into three major functional groups: receptors, transporters or enzymes. We extended this analysis by determining the membrane proteome of 24 organisms that covers all major groups of eukaryotes. This comprehensive membrane protein catalog of over 100,000 proteins was utilized to determine the evolutionary history of all membrane protein families throughout eukaryotes.  We also investigated the evolutionary history across eukaryotes of the antiviral Interferon induced transmembrane proteins (IFITM) and the G protein-coupled receptor (GPCR) superfamily in detail.  We identified ten novel human homologs to the IFITM proteins, which together with the known IFITMs forms a family that we call the Dispanins. Using phylogenetic analysis we show that the Dispanins first emerged in eukaryotes in a common ancestor of choanoflagellates and animals, and that the family later expanded in vertebrates into four subfamilies. The GPCR superfamily was mined across eukaryotic species and we present evidence for a common origin for four of the five main human GPCR families; Rhodopsin, Frizzled, Adhesion and Secretin in the cAMP receptor family that was found in non-metazoans and invertebrates, but has been lost in vertebrates. Here we present the first accurate estimation of the human proteome together with comprehensive functional and evolutionary classification and extend it to organisms that represents all major eukaryotic groups. Moreover, we identify a novel protein family, the Dispanins, which has an evolutionary history that has been formed by horizontal gene transfer from bacteria followed by expansions in the animal lineage. We also study the evolution of the GPCR superfamily throughout eukaryotic evolution and provide a comprehensive model of the evolution and relationship of these receptors.

Includes bibliography. / iv, 87 leaves, [28] leaves of plates : ill. ; 31 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1986

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.